Optical imaging and microscopy has almost four centuries of history with new developments being engineered continuously. Optical imaging operates on contrast mechanisms that offer highly versatile ability to visualize cellular and sub-cellular function and structure of biological objects under investigation. Correspondingly, optical microscopy and imaging are overwhelmingly utilized in biomedical research, for example in immunohistochemistry, in-vitro assays or cellular imaging in-vivo. The compelling advantages of fluorescence are reflected on the recent development of powerful classes of fluorescent tags that can stain functional and molecular processes in-vivo.
A large variety of optical microscopy approaches exist. As an example, in a wide-field fluorescence microscope, the entire specimen is flooded evenly in light from a light source. All parts of the specimen in the optical path are excited at the same time and the resulting fluorescence is detected by the microscope's photo detector or camera including a large unfocused background part, which does not allow three-dimensional imaging. In contrast, advanced methods, such as confocal or multi-photon microscopes, use instead a point illumination to attain three-dimensional tissue sectioning capability. For instance, U.S. Pat. No. 3,013,467 teaches on a confocal microscopy apparatus, which uses a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus signal. Multi-photon microscopy achieves a similar effect by selective detection of multi-photon absorption processes occurring in the optical focus. As only light produced by fluorescence very close to the focal plane can be detected, the spatial resolution, particularly in the sample depth direction, is therefore much better for focused methods compared to wide-field microscopes. Since in scattering tissues light can only be focused at a limited depth, microscopic observations are usually limited to specimen or depths of a few tens to a few hundreds of microns.
A major disadvantage of optical microscopy is its dependence on tissue scattering that does not allow imaging beyond a few hundred microns of fully diffusive tissue. This allows only imaging of superficial events that can be misleading and often does not allow interrogation of deeper seated structures. The depth limitations of optical microscopy have shaped entire segments of biology. Even with the advent of confocal and multi-photon tissue sectioning microscopy, only sub-millimeter tissue sections can generally be visualized due to photon scattering by tissue. Imaging deeper in tissue requires the use of (semi-) transparent specimen, i.e only in a few-day old fish or worms. For imaging deeper, the use of implanted windows in mice has been suggested, but even then, only the first few hundred of microns of disease can be visualized.
Optical Projection Tomography (OPT) being the optical analogue to X-ray computed tomography, is a different microscopic method for tomographic imaging of larger objects, which is based on the projection of light through whole transparent specimen.
U.S. Pat. No. 8,014,063 describes an OPT system, which utilizes sample rotation for acquiring transillumination tomographic data. Single plane illumination microscopy (SPIM), described in US 2007/0109633, teaches a similar method, whereby optical sectioning capability is provided by specimen illumination with a thin light sheet. Fluorescence detection is then performed at an angle of 90° relative to the illumination axis.
Larger objects can in principle be imaged with OPT, however the method can be applied in-vivo only to transparent organisms or else to chemically treated specimen to optically “clear” the tissue. This aspect limits the application to mainly fixed specimens as the treatment is toxic. Additionally, the performance of the technique depends on the penetration ability of the chemicals into the tissue and the effectiveness of photon scattering reduction. Similarly to other methods based on optical detection, also SPIM cannot penetrate more than a few hundred microns of fully scattering tissue.
In summary, in current optical microscopy systems and methods the depth that can be achieved is limited. Therefore, in the biological and medical field optical microscopy is limited to superficial tissue investigations only.
Opto-acoustic imaging is a further growing field of imaging techniques that has now seen different implementations, including the multi-spectral optoacoustic tomography approach, which enables imaging based on contrast mechanisms similar to optical microscopy, such as fluorochromes and fluorescent proteins, intrinsic cellular chromophores and extrinsically administered probes and nanoparticles. Opto-acoustic imaging has demonstrated powerful performance in imaging of optical contrast deep within several centimeters of living tissues. This is because opto-acoustics can visualize optical absorption with resolution that is not affected by photon scattering. Instead, optical contrast and markers are visualized with ultrasonic resolution. Using advanced illumination systems, high frequency detectors, and reconstruction methods, the currently achieved opto-acoustic imaging quality has already shown high potential for high-resolution imaging within several millimeters of fully scattering tissue.
Opto-acoustic imaging is insensitive to tissue scattering and can extend the application regime of optical microscopy deeper in tissues with relatively high (mesoscopic) resolution. Yet, despite significant progress, the current mesoscopic opto-acoustic imaging implementations are still limited in terms of achievable spatial resolution. Therefore, specimen of less than 5 to 7 mm in diameter could not be imaged with good image quality. Scanning times could reach an hour or more for three-dimensional imaging. Sample placement required experience and was complicated by insufficient holders. Also no optical view of the specimen was available during scanning, which further complicated specimen placement.
Zhang et al. (“Opt. Express” vol. 18, 2010, p. 1278-1282, U.S. Pat. No. 8,016,419 B2, and US 2010/0249562 A1) have proposed a combination of confocal microscopy and opto-acoustic imaging. According to Zhang et al., both of an optical image and an opto-acoustic image of an object are reconstructed. The optical image is collected with a confocal microscopy set-up, while the opto-acoustic image is collected with a single detector element or a detector array. The confocal microscopy set-up inherently requires a focussed illumination of the object. Due to the scattering properties of biological objects, the focussed illumination is restricted to the surface section of the object. The opto-acoustic image obtained by Zhang et al. is restricted to the surface section of the object, as well. The opto-acoustic image is restricted to the region, where the focussed illumination is provided.